PROJECT SUMMARY/ABSTRACT Prostate cancer (PC) is the most frequently diagnosed male cancer and second leading cause of male cancer death. Inhibiting activity of the androgen receptor (AR) is the primary treatment modality for relapsed or metastatic PC. Invariably, PC recurs with a lethal castration-resistant (CR) phenotype that is resistant to AR- targeted therapies. Clinical and experimental evidence supports the current paradigm that AR reactivation is a key driver of this resistance, supporting continued growth and progression of CRPC. This knowledge has led to the development of new therapies that inhibit AR reactivation in CRPC, but resistance remains a persistent problem for patients. The long-term objectives of this research are to determine the mechanisms that can support persistent AR transcriptional activity despite AR-targeted therapy, and to develop new strategies for treating patients with CRPC. The central discovery driving this project is a high frequency of structural rearrangements affecting the architecture of the AR gene, specifically in tumors obtained from CRPC-stage patients. The major challenge is the diversity and heterogeneity of these AR gene rearrangements, on both an intra-patient and an inter-patient basis, which poses a barrier to developing a mechanistic understanding of their clinical significance and therapeutic targeting. Preliminary data presented in the proposal show that diverse AR gene rearrangements impart a growth advantage for CRPC cells grown under the selective pressure of AR-targeted therapies, indicating that they play a central, albeit poorly-understood, role in resistance. Preliminary data also show that a common molecular outcome of these AR gene rearrangements is synthesis of truncated AR variants lacking the AR ligand binding domain. These AR variant species are able to support persistent AR transcriptional activity through mechanisms that are constitutive, ligand independent, and antiandrogen resistant. The overarching goals of this project are to understand the generality with which this new resistance mechanism occurs in CRPC-stage tumors and identify new therapeutic targets. To achieve these goals, we will develop new genomics technologies that will enable accurate identification and reconstruction of the rearranged AR gene architectures occurring in the tumors of CRPC patients. We will also use genome engineering approaches to understand whether diverse AR gene rearrangements are functionally equivalent in driving resistance to AR-targeted therapy, and understanding the necessity and sufficiency of AR variants for promoting this phenotype. Finally, the molecular mechanisms by which AR variants bind chromatin and achieve constitutive transcriptional activity will be elucidated, with emphasis on co-regulators that support these activities. These co-regulators will be evaluated as targets for inhibiting activity of AR variants downstream of AR gene rearrangements. Overall, success with these studies will elucidate the role of a frequent yet unexplored class of alteration impacting the AR gene in CRPC-stage tumors and identify new molecular targets for inhibiting AR reactivation in CRPC.